Protein translocation and folding are facilitated in the cell by helper proteins known as molecular chaperones. The heat shock protein 70 family (hsp70) of molecular chaperones is central to these processes. The goal of this research proposal is to elucidate the functions of a newly discovered chaperone called Cer1p, found in the endoplasmic reticulum (ER). Cer1p has limited homology with known hsp70s and may have some overlapping functions with the ER hsp70, Kar2p. Cer1p, like Kar2p, is important for translocation, though only for those proteins being translocated posttranslationally. A combination of genetic, molecular biological, and biochemical approaches will be employed to investigate the role of Cer1p in translocation and its restriction to a subset of the import routes. By generating rapid onset mutations long term adaptation to loss of Cer1p can be eliminated, and the direct functions of Cer1p can more conclusively be revealed. Multiple strategies are proposed to generate rapid onset mutations of cer1. Several approaches will be used to investigate the molecular basis for Cer1p's restriction to the posttranslational import pathway. Biochemical fractionation of detergent extracts will be performed to identify potential interacting partners with Cer1p and to determine if these interactions are pathway specific. Whether Cer1p directly interacts with translocating chains will be determined by crosslinking assays using microsomes. Initial pulse chase experiments suggest that Cer1p plays a role in protein folding. Funds are requested to complete a series of in vivo folding experiments to assess the relative contributions of Cer1p, Kar2p, and the hsp40 homolog Scj1p in protein folding in the ER. In addition, biochemical experiments with purified components are proposed to directly compare the properities and regulation of Cer1p and the true ER hsp70, Kar2p. Together, these studies are designed to investigate how the activities of these related chaperones are controlled to function in the translation and folding pathway in the ER.